Abstract As molecular diagnostics approaches to pathogen detection and characterization increase and become more widespread, there is increased need for efficient, consistent nucleotide extraction. In larger laboratories this is often accomplished by using automated equipment allowing for consistent results and decreasing required technician time. This proposal will compare the manual extraction with that of a smaller, less expensive, nucleotide extraction system that uses the same procedures and reagents as the standardized KingFisher Flex used in NAHLN laboratories. The KingFisher Duo Prime will be used to extract genomic DNA from ATCC strains of example gram positive (Listeria monocytogenes) and gram negative (Salmonella enterica serotype Typhimurium) bacteria as well as RNA and DNA viruses. Key Project Areas 1. Validation of automated nucleotide extraction system: Virginia Tech Animal Laboratory Services (ViTALS) has been sequencing animal specimens for pathogen detection and characterization for 4 years since entry in the MinION `Early Access Program'. We have used the MinION and iSeq for whole genome sequencing of bacteria and viruses using hybrid assembly for improved coverage and accuracy. We have the expertise to evaluate the quality of DNA and RNA extracted using quantification by Qubit III fluorometer and quality by Agilent Tapestation and finally by evaluating the quality of the subsequent sequencing. 2. Participation in additional projects: ViTALS has the intellectual infrastructure in place to conduct method development and validation as well as matrix extension as needed. If funding is received for this project, funds will be used enhance molecular diagnostic testing capabilities and are excited to investigate disease outbreaks as requested.